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Title Distribution of Lutzomyia ayacuchensis, the vector of Andean-type cutaneous leishmaniasis, at different altitudes on theAndean slope of Ecuador

Author(s) Gomez, Eduardo A.; Kato, Hirotomo; Mimori, Tatsuyuki; Hashiguchi, Yoshihisa

Citation Acta Tropica, 137, 118-122https://doi.org/10.1016/j.actatropica.2014.05.006

Issue Date 2014-09

Doc URL http://hdl.handle.net/2115/57012

Type article (author version)

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File Information ActaTropica_137p.118.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Distribution of Lutzomyia ayacuchensis, the vector of Andean-type cutaneous

leishmaniasis, at different altitudes on the Andean slope of Ecuador

Eduardo A. Gomeza, Hirotomo Katob*, Tatsuyuki Mimoric, Yoshihisa Hashiguchid,e,f

aDepartamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica de

Guayaquil, Ecuador

bLaboratory of Parasitology, Department of Disease Control, Graduate School of

Veterinary Medicine, Hokkaido University, Japan

cDepartment of Microbiology, Faculty of Life Sciences, Graduate School of Health

Sciences, Kumamoto University, Japan

dCentro de Biomedicina, Universidad Central del Ecuador, Ecuador

ePrometeo, Secretaría Nacional de Educacion Superior, Ciencia, Tecnologia e

Innovacion (SENESCYT), Ecuador

fDepartment of Parasitology, Kochi Medical School, Kochi University, Japan

*Corresponding author at: Laboratory of Parasitology, Department of Disease Control,

Graduate School of Veterinary Medicine, Hokkaido University, North 18 West 9,

Kita-ku, Sapporo, Hokkaido, 060-0818, Japan.

Phone & Fax: +81-11-706-5196.

E-mail address: hkato@vetmed.hokudai.ac.jp

Abstract

Distribution of the vector species is a major risk factor for the endemicity of

leishmaniasis. In the present study, the vertical distribution of Lutzomyia (Lu.)

ayacuchensis, the vector of Leishmania (Leishmania) mexicana in the Ecuadorian

Andes was surveyed at different altitudes (300-2,500 m above sea level) of the Andean

slope. The vector species Lu. ayacuchensis was identified at an altitude of 650 m and a

higher areas, and higher distribution ratio of the species was observed at higher altitudes.

In addition, high ratios of L. (L.) mexicana infection were detected in higher areas, but

none in lower populations of sand flies. Since an association between sand fly

populations and vector competence is suggested in Lu. ayacuchensis, haplotype analysis

was performed on the species from different altitudes of the study areas; however, no

apparent difference was observed among populations. These results suggested that Lu.

ayacuchensis in Andean slope areas of Ecuador has the potential to transmit L. (L.)

mexicana and spread leishmaniasis in these areas.

Keywords: Phlebotomine sand fly; Andean slope; Lutzomyia ayacuchensis; Leishmania

(Leishmania) mexicana; Ecuador

1. Introduction

Leishmaniasis is a vector-borne parasitic disease caused by an obligate intracellular

protozoan of the genus Leishmania. The disease is one of the most neglected diseases

worldwide, having strong and complex associations with poverty, and it affects at least

12 million people (Alvar et al., 2012). Approximately 20 species of Leishmania are

reported to be pathogenic to humans, and the parasites produce a wide range of clinical

infections in both humans and vertebrate animals as zoonoses. In humans, the disease

occurs in three distinct manifestations, cutaneous, mucocutaneous and visceral forms,

and these clinical forms are largely associated with the Leishmania species responsible.

The parasites are transmitted by female sand flies of the genus Phlebotomus in the Old

World and Lutzomyia in the New World. At present, approximately 800 sand fly species

have been recorded; however, less than 10% of them transmit each particular

Leishmania species (Munstermann, 2004; Bates, 2007; Kato et al., 2010). Therefore,

investigations on the prevalent parasite and vector species at given endemic areas is

important for risk assessment and appropriate treatment of the disease.

Since 1982, we have been conducting epidemiological studies in the New World,

especially in Ecuador. In this country, transmission of cutaneous and mucocutaneous

leishmaniases occurs in rural populations living in bilateral regions of the Andes

Mountains from the lowlands to the highlands up to an elevation of 2,500 m. The

disease is widely spread in most provinces and is a considerable public health problem

in Ecuador (Hashiguchi and Gomez, 1991). In the endemic areas, however, adequate

epidemiological studies have not been done on a community basis, and no control

measures have been applied to reduce or interrupt the transmission of the disease.

During our research activities, we have discovered a form of the disease in people

living on the Andean plateau/valleys; the disease form is very similar to ‘Peruvian uta’

but the causative agent and vector are completely different (Takaoka et al., 1990;

Hashiguchi et at., 1991; Gomez and Hashiguchi, 1991; Kato et al., 2005, 2008a). These

findings have increased the known distribution of Andean leishmaniasis in the Andes

regions. Until very recently, the only form of leishmaniasis in the Andes was thought to

be ‘Peruvian uta’ caused by Leishmania (Viannia) peruviana. In summary, in Peru the

causative agent of the disease is L. (V.) peruviana and the suspected vectors are

Lutzomyia (Lu.) verrucarum, Lu. peruensis, and Lu. ayacuchensis (Davies et al., 1993;

Perez et al., 1994, 2007; Caceres et al., 2004; Kato et al., 2008a, 2011). In Ecuador,

however, two species of the genus Leishmania, Leishmania (Leishmania) mexicana and

L. (L.) major-like, seemed to be involved in Andean highland leishmaniasis (Hashiguchi

et al., 1991). The only incriminated vector species is Lu. ayacuchensis, and the ratio

infected by L. (L.) mexicana is high (1-8%) in highland areas, which is uncommon in

most endemic areas of Ecuador (Takaoka et al., 1990; Gomez and Hashiguchi, 1991;

Hashiguchi et al., 1991; Kato et al., 2005, 2008a). Therefore, distribution of the vector

species is a major risk factor for the endemicity of leishmaniasis. In the present study,

the vertical distribution of Lu. ayacuchensis and other man-biting sand fly species were

surveyed at different altitudes (300-2,500 m above sea level) of the Andean slope, and

the genetic divergence of Lu. ayacuchensis was analyzed by targeting the cytochrome

oxidase I gene .

2. Materials and Methods

2.1. Study sites

The main study sites are located in Chimborazo Province on the south east of

Ecuador, and located on the Andean slope, ranging from 300 m to 2,500 m above sea

level (a.s.l.) along a railway, which was recently reconstructed and restarted after an

approximately 20-year break (Fig.1). The study area Canton Alausi has a population of

ca. 4,000 and lies at 2,300-2,500 m a.s.l. (Fig. 1). A small village, Chanchan, has only

37 villagers in total, and Canton Huigra has a population of ca. 2,000 at an altitude of

1,200-1,300 m a.s.l. Vegetation in these three areas is sparse, and consists of typical

Alpine flora. Another four sites on the Andean slope, Olympo (820 m a.s.l.), Ochoa

(650 m a.s.l.), Naranjapata (500 m a.s.l.) and La Ventura (300 m a.s.l.), were also

included. In these lower areas, there are scattered human dwellings and cultivated fields

along with the railway. In the Andean highlands (Alausi, Chanchan, and Huigra), L. (L.)

mexicana is endemic and Lu. ayacuchensis is incriminated as the vector species

(Takaoka et al., 1990; Gomez and Hashiguchi, 1991; Hashiguchi et al., 1991; Kato et al.,

2005, 2008a). No Andean type of cutaneous leishmaniasis (CL) cases caused by L. (L.)

mexicana was reported in the lower areas, including Olympo, Ochoa, Naranjapata, and

Ventura. In the neighboring areas, however, cases caused by L. (V.) guyanensis were

reported from Cumanda, very close to the present study site at La Ventura (Kato et al.,

unpublished). In addition, L. (V.) guyanensis and L. (V.) panamensis are prevalent in

other areas, such as Troncal, Zhucay, Manta Real, Ocaña (Cañar Province) and Naranjal

(Guayas Province), also close to La Ventura.

2.2. Collection and identification of sand flies

Sand flies were collected mainly by human landing (protected human bait)

collections from July to August, 2012 at seven sites on the Andean slope of Ecuador;

Alausi, Chanchan, Huigra, Olympo, Ochoa, Naranjapata and La Ventura, Province of

Chimborazo. Similar surveillance was performed in 1994 in our previous study. In

addition, the man-biting activity of Lu. ayacuchensis, the vector of L. (L.) mexicana in

Ecuadorian Andes, was also examined at Alausi on three days, 3rd, 17th and 28th, of

August 2013.

The sand flies were dissected and identified based on the morphology of their

spermathecae, measurements of wing veins, the ratio of palpus length to length of

antenna and the color of the thorax (Young and Duncan, 1994). The infection of sand

flies by Leishmania promastigotes in the gut was examined under a microscope.

Dissected Lu. ayacuchensis were individually fixed in absolute ethanol and stored at

room temperature for further molecular analyses.

2.3. DNA extraction

Individual ethanol-fixed sand flies were lysed in 50 l of DNA extraction buffer

[150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA and 0.1 % sodium dodecyl

sulfate (SDS)] with 100 g/ml of proteinase K. The samples were incubated at 37˚C

overnight, heated at 95˚C for 5 min, and then, 0.5 l portions were directly used as the

templates for PCR amplification.

2.4. PCR amplification and sequence analysis of the Lutzomyia ayacuchensis

cytochrome oxidase I gene

The Lu. ayacuchensis cytochrome oxidase I (COI) gene was amplified with

universal COI primers (LCO1490: GGTCAACAAATCATAAAGATATTGG and

HCO2198: TAAACTTCAGGGTGACCAAAAAATCA) (Folmer et al., 1994). PCR

amplification was carried out in a volume of 15 μl with the primers (0.4 μM each),

Ampdirect Plus (Shimadzu Biotech, Tsukuba, Japan), and high fidelity DNA

polymerase (KOD-Plus-ver.2; TOYOBO, Tokyo, Japan). After an initial

denaturation at 95˚C for 5 min, amplification was performed with 35 cycles of

denaturation (95˚C, 1 min), annealing (55˚C, 1 min) and polymerization (72˚C, 1 min),

followed by a final extension at 72˚C for 10 min. The PCR products were purified using

a FastGene Gel/PCR Extraction kit (NIPPON Genetics, Tokyo, Japan) to remove

excessive primers, and the sequences were directly determined with a forward primer by

the dideoxy chain termination method using a BigDye Terminator v3.1 Cycle

Sequencing Kit (Applied Biosystems, Foster City, CA).

2.5. Data analysis

The sequences were aligned with CLUSTAL W software (Thompson et al., 1994)

and examined using the MEGA program (Molecular Evolutionary Genetics Analysis)

version 5.2 (Tamura et al., 2011). The haplotype analysis was performed using DnaSP

5.0 (Rozas et al., 2003), and a haplotype network was constructed using the

median-joining methods as implemented in the program NETWORK 4.6.1.2

(http://www.fluxus-engineering.com/sharenet.htm) (Bandelt et al., 1999).

3. Results

3.1. Vertical distribution of sand flies along the Andean slope

Eight man-biting species of the genus Lutzomyia, Lu. ayacuchensis, Lu.

maranonensis, Lu. robusta, Lu. hartmanni, Lu. gomezi, Lu. trapidoi, and Lu.

panamensis were identified at different altitudes/sites of the present study areas (Table 1,

Fig. 2). The vector species of Andean-type cutaneous leishmaniasis, Lu. ayacuchensis,

was identified at altitude of 650 m and higher areas, and a higher distribution ratio of

the species was observed at higher altitudes; from 23.4% in Ochoa (650 m a.s.l.) to

100% in Alausi (2,300 m a.s.l) (Fig. 2). At lower sites, Lu. gomezi, Lu. hartmanni, and

Lu. trapidoi were dominant, corresponding to our previous findings (Terayama et al.,

2008). Therefore, the habitat of Lu. ayacuchensis is considered to be areas higher

than 500 m a.s.l. in Ecuador. Similar findings were obtained in the surveillance of 1994,

showing that Lu. ayacuchensis was identified at higher altitudes (>500 m) and the

distribution ratio increased with increasing altitude (Fig. S1). In 1994, the ratio of

species distribution fluctuated and a small number of Lu. shannoni (1.1%) were

identified in Naranjapata in addition to the seven species mentioned above; however, no

clear-cut difference was observed between the two surveys. Among them, only Lu.

ayacuchensis was positive for natural infection with promastigotes in highland areas,

Alausi (2.1%) and Chanchan (2.5%) in 2012, and Alausi (5.6%), Chanchan (1.8%) and

Huigra (0.6%) in 1994, but no positive sand flies were detected in other study sites

(Table 1). The parasites were all identified as L. (L.) mexicana by multilocus enzyme

electrophoresis or cytochrome b gene analyses. The biting activity of Lu. ayacuchensis

is shown in Fig. 3 in relation to the temperature and humidity at Alausi in 2013. Sand

flies started to be captured around 17:00 before sunset, peaked between 19:00 and 19:30,

and decreased as the temperature dropped under 15 degrees centigrade (Fig. 3). Similar

observations were made in other highland areas (Chanchan and Huigra). In lower areas,

sand flies started to be captured after dark. Humidity was largely unaltered during the

collection at Alausi, and it did not seem to be a major factor influencing the biting

activity of sand flies (Fig. 3). In this trial, CDC and Shannon light traps were also tested.

Among a total collection of 435 sand flies, 426 (97.9%) by human landing collection, 9

(2.1%) by CDC trap, and none (0.0%) by Shannon trap were captured. Thus, Lu.

ayacuchensis showed an extremely high affinity for humans, but not to light traps.

3.2. Haplotype analysis of the Lutzomyia ayacuchensis COI gene

High ratios of Leishmania infection in Lu. ayacuchensis were observed in highland

areas (Alausi, Chanchan and Huigra), but not at lower sites (Table 1). Since an

association between sand fly population and vector competence is suggested in Lu.

ayacuchensis (Kato et al., unpublished), haplotype analysis was performed on the

species from areas with different altitudes targeting a 621bp-fragment of the

mitochondrial COI gene. Haplotype analysis showed that 12 flies from Alausi, 8 flies

from Chanchan, 11 flies from Huigra, 12 flies from Olympo, and 5 flies from Ochoa

belonged to 5, 5, 6, 3, and 4 haplotypes, respectively (Fig. 4A). In this analysis, a

dominant haplotype, Hap2, in which sand flies from all 5 areas were included, was

noted, but no marked genetic divergence was observed among populations (Fig. 4A and

4B). No relationship between haplotypes and vertical distribution was observed.

4. Discussion

In the present study, sand fly species were surveyed at different altitudes of an

Andean slope, and vertical distribution of Lu. ayacuchensis, the vector of L. (L.)

mexicana in the Ecuadorian Andes, was elucidated. The distribution pattern was similar

to that of 20 years ago. A haplotype analysis of Lu. ayacuchensis targeting the COI gene

showed no apparent difference among populations at different localities where the

infection ratio of the sand flies by L. (L.) mexicana is markedly different.

Cutaneous leishmaniasis (CL) caused by L. (L.) mexicana is endemic in Andean

highland areas of southern Ecuador such as Huigra, Chanchan, Alausi, and Paute

(Hashiguchi and Gomez, 1991). On the other hand, L. (V.) guyanensis and L. (V.)

panamensis are widely prevalent in lowland subtropical regions, including areas close

to La Ventura (Kato et al., unpublished). Surveillance of sand flies throughout the

Andean slope identified the vertical distribution of the prevalent species; Lu.

ayacuchensis at higher areas (>650 m a.s.l.), Lu. maranonensis, and Lu. robusta on the

Andean slope (approximately 500-1,500 m a.s.l.), and L. hartmanni, Lu. gomezi, Lu.

trapidoi, and Lu. panamensis at lower subtropical areas (

L. (V.) guyanensis and L. (V.) panamensis, respectively, in subtropical areas (Momori et

al., unpublished), and Lu. tortura as a vector of L. (V.) naiffi in Amazonian areas (Kato

et al., 2008b, 2013). To date, no sand flies infected by Leishmania species have been

detected at subtropical study sites, and their vector competence has not been elucidated.

Therefore, further vector research will be needed to understand the transmission

mechanism of leishmaniasis. Lutzomyia gomezi and Lu. trapdoi, dominant species in

Naranjapata and Ventura, may have the potential to spread leishmaniasis as reported in

other areas.

Characteristically, the infection rate of Lu. ayacuchensis by L. (L.) mexicana is

constantly high (1-8%) in the Ecuadorian Andes although the ratio in sand flies is

mostly less than 1% in subtropical areas (Takaoka et al., 1990; Gomez and Hashiguchi,

1991; Hashiguchi et al., 1991; Kato et al., 2005, 2008a). Although Lu. ayacuchensis is

circulating in Andean “slope” areas (Olympo and Ochoa), infection of the species by

Leishmania was not found in the present or past studies. In addition, no CL case caused

by L. (L.) mexicana has been reported in these areas. These observations raise the

possibility that vector competence may be different between the Andean “highland” and

“slope” populations, since our recent study suggested an apparent association between

population and vector competence in Lu. ayacuchensis (Kato et al., unpublished). A

haplotype analysis based on COI genes showed no striking difference between the

populations despite our expectations. Therefore, factors other than sand flies are

considered to contribute such a distinct infection ratio, as well as the endemicity of

leishmaniasis between Andean “highland” and “slope” areas. The presence or absence

of appropriate reservoir animals due to particular flora and fauna may cause such

differences. However, Lu. ayacuchensis of the Andean slope populations is considered

to have potential to transmit L. (L.) mexicana, and thus, the species in these areas can be

a risk factor for the expansion of leishmaniasis.

In the present study, a vertical distribution of sand flies through an Andean slope

(300-2,300 m a.s.l.) was identified, and the distribution range of Lu. ayacuchensis, the

vector of L. (L.) mexicana, was determined. In addition, no marked genetic divergence

was found in Lu. ayacuchensis populations between Andean “highland” and “slope”

areas. These results suggest that Lu. ayacuchensis in Andean “slope” areas has potential

to transmit L. (L.) mexicana, although CL caused by the parasite species, L. (L.)

mexicana, is not endemic at the present time. Continuous further surveillance of sand

flies regarding natural infection with Leishmania will be necessary in and around these

areas as they are a risk factor of the endemicity of leishmaniasis.

Funding

This study was financially supported by the Ministry of Education, Culture and

Sports, Science and Technology (MEXT) of Japan (Grant Nos. 23256002 and

25257501), and the Prometeo Project of the Secretaria Nacional de Educacion Superior,

Ciencia, Tecnologia e Innovacion (SENESCYT), Ecuador.

Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgements

We are indebted to Flavio-Valeriano Zambrano C. (Servicio Nacional de

Erradicacion de la Malaria, Guayaquil, Ecuador), Kazue Hashiguchi (Centro de

Biomedicina, Universidad Central del Ecuador, Quito, Ecuador), and Roberto Sud A.

(Ministerio de Salud Publica y Asistencia Social, Guayaquil, Ecuador) for their

technical assistance during the field phase of the present study.

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Figure Legends

Fig.1. (A) Map of Ecuador and Chimborazo Province where the main study sites are

located. Sand flies were collected at the sites on Andean slopes, ranging from 300 m to

2,500 m above sea level (a.s.l.), along a railway (broken line). (B) Elevation map of the

study areas; Alausi (2,300 m a.s.l.), Chanchan (1,500 m a.s.l.), Huigra (1,200 m a.s.l.),

Olympo (820 m a.s.l.), Ochoa (650 m a.s.l.), Naranjapata (500 m a.s.l.) and La Ventura

(300 m a.s.l.).

Fig.2. The proportion of sand fly species captured at each study site from July to August

in 2012. AL, Alausi; CH, Chanchan; HU, Huigra; OL, Olympo; OC, Ochoa; NA,

Naranjapata; VE, La Ventura.

Fig.3. Man-biting activity of Lutzomyia ayacuchensis at Alausi on three days (3rd, 17th

and 28th, of August 2013). Horizontal axis indicates collection time. Line plots show

average temperature (●) and humidity (▲), and bar graphs and the numbers above the

bars indicate the number of sand flies collected during each collection period.

Fig.4. (A) Variable nucleotides found in the alignment of the Lutzomyia (Lu.)

ayacuchensis cytochrome oxidase I (COI) gene. The COI gene sequence of a

621bp-fragment was analyzed in 48 Lu. ayacuchensis collected from 5 sites (AL,

Alausi; CH, Chanchan; HU, Huigra; OL, Olympo; OC, Ochoa). Dots denote identical

sequences and numbers show their corresponding positions from a Lu. ayacuchensis

COI gene fragment analyzed in this study. (B) Haplotype network of the COI sequences

of Lu. ayacuchensis collected from 5 study sites. Each haplotype is represented by a

circle sized in proportion to the frequency of the haplotype. Each crossbar represents

one nucleotide substitution.

Fig.S1. The proportion of sand fly species captured at each study sites in 1994. AL,

Alausi; CH, Chanchan; HU, Huigra; OL, Olympo; OC, Ochoa; NA, Naranjapata; VE,

La Ventura.